MRP1 and MRP2, the founding members of the MRP family of ABC transporters, are drug efflux pumps that play prominent roles in cellular resistance to anticancer agents and in drug distribution. These two pumps are now known to be part of a multigene family that extends to 9 members. Recent studies in my laboratory and others have determined that the first three of the newly identified family members to be examined, MRP3, MRP4 and MRP5, are also drug pumps involved in cellular resistance to anticancer agents. MRP3 is a glutathione and glucuronate conjugate efflux pump that has the facility for conferring resistance to etoposide and methotrexate, and may also contribute to the oral bioavailability of these agents, as well as participate in the defense of hepatocytes during cholestatic conditions that prevent these agents from being extruded into the bile. MRP4 and MRP5 are cyclic nucleotide efflux pumps that are deployed by the cell as resistance factors for nucleotide analogs such as 6-mercaptopurine, and the resistance profile of MRP4 includes methotrexate as well. These recent developments have disclosed that at least 3 of the new members of this family are involved in important areas that relate directly to cancer chemotherapeutic agents, and provide a compelling reason why the activities of each of the newly identified members of this family should be investigated in depth. In this competitive renewal we will extend our previous investigation of MRP4 and continue our analysis of other newly identified family members. With regard to MRP4, we will determine the biochemical mechanism by which it confers resistance to nucleotide analogs, further explore its potential for conferring resistance to cancer chemotherapeutic agents, and assess its in vivo potency as a resistance factor by the use of experimental mouse models. In addition, we will analyze the drug resistance capabilities and substrates selectivities of two of the newly identified family members whose structural resemblance to MRP4 and MRP5 suggests conserved or overlapping functions and whose investigation may synergize with our concurrent studies on MRP4. These experiments should provide a more complete picture of the potential for this complex family of unusual pumps to impact cancer treatment.